Identification of Fodrin Protein in Postsynaptic as a Major Calmodulin-binding Density Preparations

A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X100, has been found to be identical to fodrin (Levine, l., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-12Sl-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido-calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin. In our initial paper (13) on the isolation and characterization of postsynaptic densities (PSD) from canine cerebral cortex, we observed a doublet band on denaturing gels of the proteins of the PSD. We pointed out then (13) that this doublet (which we then labeled as Mr 185,000 but have since corrected to M~ 230,000) seems to be an intrinsic protein of the PSD, since it is concentrated in the PSD fraction over that of a synaptic membrane fraction from which the PSD was derived (11, 13). Further evidence for its being intrinsic to the PSD is that the doublet resists solubilization by the ionic detergents, deoxycholate (0.5%) and sarkosyl (1.0%) (31), and that it is also found in the PSD fraction isolated from cerebellum (9). Further work (11) on calmodulin-binding proteins in the PSD showed, by a gel-overlay technique, that this protein binds calmodulin, though at that time we could not tell whether one or both of the bands bound calmodulin. Recently, Davies and Klee (14) purified by calmodulin affinity chromatography a protein from whole brain, which they named CBP-I, and which was found to also bind actin, by sedimentation analysis (14). A somewhat similar result was reported in abstract form by Beach et al. (7), in that a protein doublet, of Mr 245,000, isolated from whole brain, was found to bind actin, and to be a component of the synaptic junction complex. Willard and co-workers (25, 35) had previously shown that among the axonally transported proteins was a doublet of Mr 240,000. They have now purified this protein from whole brain (23) and found that it binds actin by sedimentation analysis. Because they localized it to the cytoplasmic surface of many cell types (23), using immunohistochemistry, they have called it fodrin, from the Greek word for lining, "fodros." Because there is evidence, presented in the Discussion, that the two proteins of this doublet are subunits of fodrin, we have denoted, for convenience, the fodrin doublet as alphaand beta-fodrin. We present evidence here that a doublet protein of Mr 230,000 and 235,000, which is concentrated in a PSD fraction, is identical to fodrin. It is instructive that both of the proteins known to bind to fodrin, actin (8, 21) and calmodulin (19), have been found to be in the isolated